D-mannose agarose beads (Sigma-Aldrich Chemie GmbH, Munich, Germany) and sepharose beads (CL-4B, ø 40 μm to 165 μm, Sigma-Aldrich Chemie GmbH, Munich, Germany) were each diluted in a ratio of 1:10 in PBS and subsequently centrifuged at 1000 rpm for 5 min. The supernatant was removed and the washing procedure was repeated twice. Finally, the cleaned beads were diluted 1:10 in PBS. 3 × 105A. castellanii or A. comandoni in 5 ml of PYG medium were seeded into tissue culture bottles (25 ml, Sarstedt AG & Co., Nümbrecht, Germany) and 0.5 ml of one of the bead solutions were added. The samples were observed with an inverted phase contrast microscope (Olympus CKX41) equipped with a camera (C9300, Hamamatsu, Hamamatsu, Japan) and 50 images of each bottle were taken right after the addition, after 2 h and after 4 h incubation time at room temperature. The number of acanthamoebae adhering to the beads and the total number of acanthamoebae in the resulting images were counted.
Cl 4b
Manufactured by Merck Group
Sourced in Germany, United States
CL-4B is a gel filtration chromatography medium used for the separation and purification of proteins, peptides, and other biomolecules. It is composed of cross-linked agarose beads with a fractionation range suitable for the separation of molecules with a molecular weight range of 1,000 to 300,000 daltons.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using cl 4b
1
Acanthamoeba Adhesion to Functionalized Beads
2
Lipid-Based Nanoparticle Delivery of siRNA
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d -Galactosyl ceramide, PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (ammonium salt)), cholesterol, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), rhodamine-conjugated DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and DOTAP (dioleoyl-3-trimethylammonium-propane) were purchase from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). The cationic lipid DMKE (O,O’-dimyristyl-N-lysyl glutamate) was synthesized as previously reported [32 (link)]. EX-CyTox that is cell viability and cytotoxicity test reagent is Daeil Lab Service Co., Ltd (Seoul, South Korea). Doxorubicin and CL-4B (agarose beads) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). FITC-labeled siRNA was purchased from Invitrogen™ Life Technologies (Grand Island, NY, USA). siRNA against vimentin was obtained from Santa Cruz biotechnology (Dallas, TX, USA).
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3
Coimmunoprecipitation of Vpu and CD47
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For coimmunoprecipitation studies of Vpu and CD47, transfected HEK 293T cells were lysed in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) buffer (50 mM Tris, 5 mM EDTA, 100 mM NaCl, 0.5% CHAPS, pH 7.2) supplemented with protease inhibitors. Lysates were first precleared by incubation with 40 μl of protein A-Sepharose beads CL-4B (Sigma, GE17-0963-03) for 1 h at 4°C and incubated with mouse MAb anti-HA (clone 16B12) overnight. The following day, 40 μl of beads was added, and samples were incubated for 2 h, washed five times with CHAPS buffer, and analyzed by Western blotting.
4
Co-Immunoprecipitation Protocol for Protein Analysis
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For co-immunoprecipitation, the cells were lysed in 1 mL 1% NP-40 lysis buffer(1% NP-40, 50 mM Tris-base, 150 mM NaCl). Cell lysates were gently shaken with 100 µL CL-4B (Sigma-Aldrich, St Louis, MO) at 4°C for 1 hr, then centrifuged at 15000 r/min 4°C for 15 min. The immunoprecipitation antibody recruited with 20 µL protein A/G (Santa Cruz Biotechnology, Santa Cruz, CA) were mixed with the supernatant and shaken at 4°C overnight. The protein-agarose beads mixture was washed once by 1% NP-40 lysis buffer and twice by ice-cold PBS, boiled with 4×SDS sample buffer, then analyzed on 10% glycine SDS-PAGE gel.
5
Co-immunoprecipitation of Vpu and CD47
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For co-immunoprecipitation studies of Vpu and CD47, transfected HEK 293T cells were lysed in CHAPS buffer (50 mM Tris, 5 mM EDTA, 100 mM NaCl, 0.5% CHAPS, pH7.
supplemented with protease inhibitors. Lysates were first precleared by incubation with 40 µL of protein A-sepharose beads CL-4B (Sigma, #GE17-0963-03) for 1 h at 4°C and then incubated with mouse mAb anti-HA (clone 16B12) overnight. The following day, 40 µL of beads were added and samples were incubated for 2 h, washed five times with CHAPS buffer and analyzed by Western blotting.
supplemented with protease inhibitors. Lysates were first precleared by incubation with 40 µL of protein A-sepharose beads CL-4B (Sigma, #GE17-0963-03) for 1 h at 4°C and then incubated with mouse mAb anti-HA (clone 16B12) overnight. The following day, 40 µL of beads were added and samples were incubated for 2 h, washed five times with CHAPS buffer and analyzed by Western blotting.
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